Characterization of the sigD transcription unit of Bacillus subtilis

Abstract

The 0FD factor of BaciUlus subtilis is required for the transcription of the flagellin and motility genes as well as for wild-type chemotaxis. Southern blot and sequence analyses demonstrate that the structural gene for SFD, sigD, is located immediately downstream of a region of DNA originally identified as the chemotaxis (che) locus and now renamed thefla/che region. In fact, sigD appears to be part of a very large operon (>26 kb) containing genes which encode structural proteins that form the hook-basal body complex as well as regulatory proteins required for chemotaxis. Transposon insertions up to 24 kb upstream of sigD, within several of the genes for the hook-basal body components, give rise to only a moderate decrease in sigD expression. The transposon insertions, however, block CrD activity as demonstrated by the lack of flagellin expression in strains bearing these insertions. These effects appear to arise from two types of regulation. In cis the transposon insertions appear to introduce a partial block to transcription of sigD from upstream promoter elements; in trans they disrupt genes whose gene products are required for oD activity. It appears that sigD transcription is initiated, at least in part, by a promoter many kilobases upstream of its translation start site and that transcription of the flagellin gene by SFD is dependent on the formation of a functional hook-basal body complex. The possibility that sigD is part of the Ia/che operon was further tested by the integration of an insertion plasmid, containing strong transcription terminators, 1.6 and 24 kb upstream of the sigD gene. In both cases, the introduction of the terminators resulted in a greater decrease of sigD expression than was caused by the plasmid sequences alone. These results indicate that wild-type transcription of sigD is dependent on promoter sequences >24 kb upstream of its structural gene and that the entirefla/che region forms a single operon.

Identification of flagellar synthesis regulatory and structural genes in a sigma D-dependent operon of Bacillus subtilis

“…This and previous studies make it clear that in B. subtilis the repression of late flagellar gene expression by a mutation in an early flagellar gene is a result of inactivation of aD and not due directly to changes in its cellular levels (43 We also envision the use of the flgMA80 mutant as a potential means to distinguish true lyt mutants from those which are Lyt-because they are fla mutants. In the latter case, the added presence of theflgMA80 mutation should result in a reversion to Lyt+, as we have seen for strain CB150.…”

Transcription of Bacillus subtilis degR is sigma D dependent and suppressed by multicopy proB through sigma D

“…The 817-bp DNA fragment containing the entire sigD-coding region was prepared by PCR amplification using the CU741 chromosomal DNA and primers sgd-1 and sgd-2. The 189-bp Sau3AI-HindIII DNA fragment that was originated from an internal part of the sigD-coding region and was used for sigD disruption by Helmann et al (5,13) was cloned into BamHIand HindIII-digested pUKM504.…”

Effect of degS-degU mutations on the expression of sigD, encoding an alternative sigma factor, and autolysin operon of Bacillus subtilis

“…1). Marquez et al (20) and Marquez-Magana and Chamberlin (21) reported that sigD may be part of a large operon containing several genes upstream of the sigD gene, and they showed that an upstream integrant in the sigD locus produced 7% of the wild-type level of the SigD protein and exhibited the synthesis of 64% of the wild-type flagellin and a decrease in swarm size (55% ± 10% of the wild-type level). Thus, the disruption of the degU gene and deficiency in DegU phosphorylation may not significantly affect motility, as other investigators had noted (24).…”